Production of glucose isomerase by bacillus coagulans

ABSTRACT

A superior glucose isomerase is present intracellularly in an atypical B. coagulans characterized by an ability to grow on inorganic sources of nitrogen.

The invention relates to glucose isomerase, to methods for theproduction of this enzyme and to enzymatic isomerization of glucose tofructose with glucose isomerase.

Syrups containing a mixture of glucose and fructose are widely used inindustry because of their sweet taste and their low tendency tocrystallize. Such syrups are preferably produced from glucose syrupsusing an enzyme to catalyze the isomerization of glucose to fructose. Itis important for the economy of this process that the enzyme costs arelow and that there be a negligible formation of by-products, which willhave to be removed before the syrup can be used.

Enzymes capable of isomerizing glucose to fructose may be obtained froma large number of different microorganism species, and the properties ofthe enzymes vary from species to species. Enzyme properties, which areparticularly important for the use in the isomerization process are,stability at high temperatures and activity at low pH values.

In an enzymatic isomerization of glucose to fructose, the followingconditions are of importance:

PH: A pH value as low as possible is desired in order to avoid thealkali-catalyzed by-product formation, i.e. below pH 7, pH 5-7 is asuitable range.

Temperature: The enzyme should be stable at a temperature of about 55°Cto 75°C, which is the usual reaction temperature.

Solubility: In many cases it is desired to use the microorganism cellscontaining the enzyme intracellularly for the isomerization; it istherefore very important that as little as possible of the enzyme leaksout in the reaction mixture during isomerization or during handling ofthe cells after isomerization. It is furthermore of importance in theproduction of the enzyme preparation that the cells easily may be driedin such a way that the enzyme will not leak out of the cells.

In other methods soluble enzyme is desired. This may be used eitherdirectly, or it may be made insoluble by coupling to an insolublecarrier. For this process it is important that the enzyme can be easilyextracted from the cells.

As the price margin between glucose and isomerized glucose is verysmall, it is furthermore extremely important that the production yieldof the enzyme is high, so that the price of the enzyme preparation canbe sufficiently low.

It has now been found that by using the isolation method according tothis invention, it will be possible reproducibly and with high frequencyto isolate organisms from nature, which will be able to produce aglucose isomerase, which has extremely good heat stability and a verylow pH optimum.

The organisms isolated in this way are aerobic sporeforming rod-shapedbacteria. They must be classified as a thermophilic Bacillus species,and more specifically are identifiable as an atypical B. coagulans,differentiated from typical B. coagulans strains by an ability to growat 65° C and to grow on inorganic sources of nitrogen.

The isolation method according to the invention consists in incubatingsources of microorganisms, e.g. soil samples, on media containing onlyammonia as nitrogen source, xylose as carbon source and the usual traceelements as inorganic salts. The incubation must take place at atemperature above 60°C, preferably between 60° and 65°C, to ensuregrowth only of thermophilic microorganisms. Agar in a concentration of1.5-3% may be added to the medium in order to make the isolation of thedeveloping organisms simpler. The organisms developing on this mediumare purified by plating on agar medium of the composition describedabove and at the same temperature. The pure cultures thus obtained arethen tested for glucose isomerase activity using a simplesemi-quantitative test, e.g. as described below in Example 1, thecultures giving a positive reaction are selected.

the rationale of the entire isolation technique is to establish growthconditions which will encourage rapid growth of microorganisms mostadapted to industrial utilization and likely to produce a glucoseisomerase.

Incubation at elevated temperatures, e.g. 60°C, encourages growth ofthermophilic microorganisms. As a class, thermophiles have the highgrowth rates desired for industrial fermentation processes. In addition,the enzymes produced by thermophiles are thermostable.

Xylose is the carbon source since all known glucose isomerases arebasically xylose isomerases. Many organisms which digest xylose willcontain a glucose isomerase. The screening therefore is an effort tolocate organisms which can use xylose as their sole source of carbon.

Ammonia, i.e. an inorganic nitrogen source, is employed to excludemicroorganisms that require organic nitrogen including notably B.coagulans, a microorganism species that would otherwise fit all theconditions of the screening program. Actually, B. coagulans is a knownsource of a quality glucose isomerase, one with relatively good heatstability and a low pH optimum for both activity and stability.Unfortunately, B. coagulans is expensive to cultivate because its growthrequirements include vitamins, and amino acids.

The microorganisms isolated by the above described screening procedurehave a number of properties in common and, it is believed, areclassifiable in a single species. However, the species in question wouldseem to be that of B. coagulans. An apparent identity between B.coagulans and the microorganisms of the present invention is notsurprising since the screening conditions are so very close toconditions which would select B. coagulans that obtaining closelyrelated microorganisms should be expected. In addition, B. coagulans isa poorly defined species with several subgroups already known to exist.The literature on B. coagulans acknowledges that a vigorousinvestigation of B. coagulans is likely to establish that B. coagulansconstitutes several different species of related microorganisms. Pendinga definitive investigation of B. coagulans, all that can be said of themicroorganism of the present invention is that it is an atypical B.coagulans characterized in its atypicality by being capable of capableof growth on inorganic sources of nitrogen.

A biochemical distinction of importance to the present invention is thatthe glucose isomerase enzyme of the atypical B. coagulans does differfrom the glucose isomerase of (typical) B. coagulans, notably withregard to thermal stability and the effect of pH and temperature onactivity. The glucose isomerase of the present invention is morethermostable and its activity at pH 6 to 7 is as good, if not better. Intotal a different enzyme is present.

For production of the glucose isomerase of the present invention, thebacteria are cultivated aerobically on media containing usual salts andsources of carbon and nitrogen at a pH between pH 5 and pH 9. Additionof xylose or xylose containing compounds to the medium is necessary inorder to induce glucose isomerase formation in the wild strains. It ishowever possible to isolate mutants which produce glucose isomerasewithout induction by xylose. When such microorganisms are cultivated,any metabolizable carbon source may be used.

Incubation temperature for the fermentation is between 40° and 65°C,usually around 50°C. Aerobic conditions are maintained during thecultivation by blowing air throgh the medium, e.g. at a rate ofapproximately one volume of air per volume of liquid per minute.

The enzyme is formed intracellularly and the purification procedureusually consists in harvesting the cells by centrifugation or filtrationand if desired drying of the cell cream by lyophilization, spray dryingor by any other process which will not destroy the enzyme activity.Before drying, the cells may be treated with solutions of Cobalt salts,so that the enzyme preparation will contain an amount of Co⁺ ⁺ necessaryto activate the enzyme.

The harvested cells may furthermore be heat-treated at a temperaturebetween approximately 70°-80°c for a time sufficient to destroy thelytic enzyme systems within the cells.

During cultivation, the amount of glucose isomerase present in the cellsmay be measured at intervals by determining the amount of fructoseformed from glucose under standard conditions. When there is no moreincrease in activity, the cells are harvested.

If a soluble enzyme product is desired, it will be possible to conductthe fermentation so that most of the enzyme leaks out of the cells intothe fermentation broth. This can for instance be done by raising thefermentation temperature to 60°C during the whole fermentation period oronly during the latter part of it.

An enzyme preparation may be prepared from this broth using the usualmethods for the preparation of extracellular enzymes, i.e. purifying thebroth by centrifugation or filtration and concentrating the enzymecontaining solution by evaporation or reverse osmosis, thus obtaining aliquid enzyme product, or by precipitating the enzyme out of the brothor the concentrated broth by means of salts or water soluble solventsand finally drying the precipitate.

A soluble enzyme may also be prepared from the isolated cells by lettingthem autolyze, by addition of lytic enzymes, e.g. lysozyme or by theaddition of surface active agents, furthermore by mechanical rupture ofthe cells such as by ultra-sonics or by the use of mechanicalhomogenizing devices. Enzyme preparations can be prepared from cellstreated in this way by the methods described above.

Normally the microorganisms will produce a number of spores during thefermentation. If a spore-free preparation is desired, an asporogenicmutant should be used for the cultivation. Methods for the isolation ofasporogenic mutants are well known in the art and it is easy to isolatethis type of mutants.

If desired, the enzyme preparation according to the invention may beimbedded into a matrix or insolubilized by any method known in the artwhich will not destroy the enzyme activity.

The present enzyme has properties which are very favorable for glucoseisomerization under the favorable commercial conditions of pH 5-7. Itmay be used from 55°-85°C without substantial loss of activity down topH values at about 5. It is extremely heat stable, retaining much of itsactivity at 85°C over the entire pH 5 to pH 7 range. By contrast, theactivity of known B. coagulans glucose isomerase loses activity withtemperatures in excess of 70°C, having almost no activity at 85°C.Superiority in properties of the enzyme make all the more advantageousthe economies achieved by cultivating the microorganism on inorganicnitrogen.

For further understanding of this invention, the following specificexamples thereof are presented.

EXAMPLE I Isolation of glucose isomerizing organisms from nature

A 10% suspension of garden soil in sterile water was spread on plateswhich contained an agar medium of the following composition in g perliter of distilled water:

    ______________________________________                                        (NH.sub.4).sub.2 SO.sub.4                                                                         3                                                         K.sub.2 HPO.sub.4   1                                                         MgSO.sub.4, 7H.sub.2 O                                                                            0.5                                                       KCl                 0.5                                                       FeSO.sub.4          0.01                                                      agar                20                                                        xylose              5                                                         ______________________________________                                    

was autoclaved separately and added aseptically just before pouring theplates.

The plates were incubated at 60°-65°C for 2 days and the colonies whichdeveloped were transferred to a medium of the following composition in gper liter of distilled water:

    ______________________________________                                        peptone             10                                                        tryptone            5                                                         yeast extract       3                                                         Na.sub.2 HPO.sub.4, 12H.sub.2 O                                                                   18                                                        MgSO.sub.4, 7H.sub.2 O                                                                            0.2                                                       KH.sub.2 PO.sub.4   7                                                         agar                20                                                        xylose              10-20                                                     ______________________________________                                    

was autoclaved separately and added aseptically just before pouring theplates.

If the culture appeared to be impure, it was restreaked on the firstmedium and again transferred to the rich medium. This procedure wasrepeated until a pure culture was obtained.

The cultures on the rich medium were incubated for 1-2 days at 60°C andwere then tested for the ability to isomerize glucose to fructose in thefollowing manner:

The cells were suspended in 0.9% NaCl. One (1) ml of cell suspension wasincubated at 70°C with 1 ml of the following solution:

    ______________________________________                                        glucose         80       grams per liter                                      TRIS            0.1      mole per liter                                       chloroform      2        ml                                                   MgSO.sub.4, 7H.sub.2 O                                                                        5        grams per liter                                      CoCl.sub.2, 6H.sub.2 O                                                                        0.5      grams per liter                                      pH was adjusted to 6.5 using HCl.                                             ______________________________________                                    

After 4 hours of incubation, one droplet of the reaction mixture wastransferred to a sheet of chromatographic paper (Whatman No. 1) by meansof a Pasteur pipette. The droplets were arranged in a manner allowingspace for 50-100 droplets per sheet of paper. After drying, the paperwas moistened in the following solution:

    ______________________________________                                        naphtalindiol 1,3     250 mg                                                  ethanol               250 ml                                                  ConcHCl                20 ml                                                  ______________________________________                                    

After drying for at least 30 minutes at room temperature the paper washeated at 60°c for 1-20 minutes. If the reaction mixture contained atleast 0.5% fructose, a brown spot became visible after this treatment.

Eighteen cultures giving positive reactions for fructose using thismethod were isolated. The cultures are deposited at the NorthernRegional Research Laboratory, Peoria, Ill., USA (NRRL) under the numbersNRRL B-5649 to NRRL B-5666.

These strains have the following properties in common:

Morphology:

Similar to that of the species in the B. subtilis group.

Biochemical reactions:

Temperature, for growth: 40°-65°C, optimum 55°-60°C. Growth inhibited byhigh concentrations of protein (above 3%) and carbohydrates (1%). Acidis produced from glucose, xylose, fructose, ribose and glucerol (no gasformed); arabinose is not fermented.

No growth in 3 % NaCl.

Growth at pH 5.7: positive.

Growth on glucose-nutrient agar better than growth on nutrient agar.Good growth on soybean agar.

Hydrolysis of casein and starch positive when tested at 40°C, weak ornegative when tested after growth at 50°C.

Negative hydrolysis of gelatine.

Anaerobic growth in glucose medium, negative (weak).

EXAMPLE II Production of glucose isomerase in shake flasks

In baffled 500 ml Erlenmeyer flasks was prepared 100 ml of a medium withthe following composition (in grams per liter tap water):

    ______________________________________                                        corn steep liquor      80                                                     yeast extract          5                                                      K.sub.2 HPO.sub.4      1                                                      (NH.sub.4).sub.2 SO.sub.4                                                                            5                                                      xylose                 4                                                      MgSO.sub.4, 7H.sub.2 O 0.2                                                    MnSO.sub.4, H.sub.2 O  0.05                                                   ______________________________________                                    

Xylose, MgSO₄ and MnSO₄ were autoclaved separately and were addedaseptically after cooling to room temperature. pH was asepticallyadjusted to 7.0 using sterilized 4N NaOH.

The flasks were inoculated with 1 ml of a suspension of cells in steriledistilled water prepared by scraping off growth from slants on the richmedium described in Example I.

The inoculated flasks were incubated on a rotary shaking table (220 rpm)at 50°C. After 40 hours of incubation, the contents of the flasks werecentrifuged and the cells obtained in the precipitate were used forisomerization of glucose in the following way:

In a reactor provided with means for maintaining a constant temperature,nitrogen atmosphere, pH control and stirring was placed 0.5 liter of a40% (w/w) solution of dextrose, H₂ O in distilled water to which wasadded 0.5 g of MgSO₄, 7H₂ O and 0.05 g of CoSO₄, H₂ O.

Glucose isomerase containing cells from 1 liter of medium were added tothe solution and the isomerization process took place under thefollowing conditions: Temperature: 80°C; pH: 6.2; Time: 10 hours.

The amount of fructose formed was determined by polarimetry and thedegree of isomerization was determined as % fructose formed/initial % ofdextrose. The following results were obtained:

    ______________________________________                                        Strain NRRL No.   % Conversion                                                ______________________________________                                        5649              35                                                          5650              44                                                          5651              42                                                          5652              44                                                          5653              47                                                          5654              44                                                          5655              50                                                          5656              42                                                          5657              42                                                          5658              38                                                          5659              38                                                          5660              42                                                          5661              42                                                          5662              51                                                          5663              42                                                          5664              44                                                          5665              42                                                          5666              47                                                          ______________________________________                                    

EXAMPLE III

Thermophilic Bacillus NRRL No. 5650 was cultivated overnight in nutrientbroth at 48°C. One ml of this culture was transferred to a 500 mlbaffled Erlenmeyer flask containing 100 ml of the following medium (ingram per liter distilled water):

    ______________________________________                                        Na.sub.2 HPO.sub.4, 2H.sub.2 O                                                                    9                                                         KH.sub.2 PO.sub.4   7                                                         MgSO.sub.4, 7H.sub.2 O                                                                            0.5                                                       KCl                 0.5                                                       FeSO.sub.4          0.01                                                      antifoam            0.1                                                       (NH.sub.4).sub.2 SO.sub.4                                                                         3                                                         xylose              5                                                         ______________________________________                                    

was autoclaved separately and added aseptically before inoculation.

The flask was incubated on a rotary shaking table at 48°C for 1-2 days.The glucose isomerizing activity was tested as described in Example II;the result was 32% conversion.

EXAMPLE IV Isolation of mutant bacteria which are able to produceglucose isomerizing activity in xylose-free media

Logarithmically growing cells in nutrient broth were harvested andsuspended in TRIS-maleate buffer pH 6.0 containing 100 micrograms ofN-methyl-N'-nitro-N-nitrosoguanidin per ml. The suspension was incubatedat 37°C for 30 minutes, whereafter the cells were washed diluted andspread on petri dishes containing nutrient agar.

After 1-2 days at 50°-60°C, colonies of mutant strains were transferredto petri dishes containing a xylose-free medium of the followingcomposition in gram per liter of distilled water:

    ______________________________________                                        yeast extract        30                                                       agar                 20                                                       ______________________________________                                    

The plates were incubated until good growth was obtained (1-2 days). Theglucose isomerase activity of the colonies was now determined using thesimple method described in Example I.

The number of mutants found was high, about one in 2,000 to 3,000colonies tested.

I claim:
 1. A process for the production of glucose isomerase,comprising cultivating under aerobic conditions an atypical Bacilluscoagulans productive of a glucose isomerase, said Bacillus coagulansbeing capable of growth on only inorganic nitrogen sources as thenitrogen source and at a temperature of 65°C on a nutrient mediumcontaining a nitrogen source, a carbon source, optionally includingxylose, small amounts of inorganic salts, at a pH value between 5 and 9and a temperature between 40° and 65°C, whereafter the glucose isomerasethus produced is recovered.
 2. A process as claimed in claim 1 in whicha mutant atypical Bacillus coagulans strain is cultivated in a growthmedium free of xylose.